More-than one percent of human IgG has a binding specificity for Gal- alpha1 ,3Gal-beta1 ,4GlcNAc. This antigen is referred to as the alpha- galactosyl epitope and the antibody is designated anti-Gal. Anti-Gal and alpha-galactosyl epitopes have a unique evolutionary distribution. Thus, humans and Old World monkeys express the antibody, but not the antigen, whereas New World monkeys and other nonprimate mammals express the antigen in abundance, but not anti-Gal. These discoveries have relevance to several clinical areas including xenotransplantation, glycoprotein drug development, autoimmunity and possibly cancer. The studies in this application address several issues concerning the enzyme (i.e. alpha1 ,3galactosyltransferase or alpha1,3GT) responsible for the synthesis of the alpha-galactosyl epitope. The proposed studies will focus on characterizing alpha1,3GT in terms of protein structure and post translational modification by electrospray and matrix-assisted laser- desorption ionization time-of-flight mass spectrometry. A novel scheme to generate a series of synthetic substrate analogs will allow a complete mapping of the key polar groups on the substrate that are recognized by the enzyme and produce a large array of potential inhibitors of the enzyme. These molecules will form the basis for a series of photoprobes that will be used to map the substrate binding site of the enzyme. Finally, molecular biological approaches will be used to determine the sequence of alpha1,3GT expressed by various mammalian species which are evolutionarily distant from each other. This will lead to the identity of a number conserved amino acids in alpha1,3GT. Some of these amino acids are likely to form the substrate binding sites and/or catalytic site of alpha1,3GT. The unique combination of scientific approaches proposed in this application will provide a significant amount of new information on alpha1,3GT, and a better understanding of glycosyltransferases in general.